Interference of urinary albumin-to-creatinine ratio measurement by glycosuria: clinical implications when using SGLT-2 inhibitors
Authors: Daniel Chapman, Parminder K. Judge, Rebecca J. Sardell, Natalie Staplin, Thomas Arnold, Doreen Zhu, Sarah Ng, Stewart Moffat, Martin J. Landray, Colin Baigent, Michael Hill, Richard Haynes, Sarah Clark, William G. Herrington
Journal: Kidney International (2023)

Urinary albumin-to-creatinine ratio (uACR) measured on spot samples provides a convenient method to screen for, diagnose, stage, and monitor chronic kidney disease (CKD),1Stevens P.E. Levin A. Evaluation and management of chronic kidney disease: synopsis of the kidney disease: improving global outcomes 2012 clinical practice guideline.Ann Intern Med. 2013; 158: 825-830Crossref PubMed Scopus (1963) Google Scholar and change in albuminuria is proposed as a surrogate outcome for CKD progression in clinical trials.2Heerspink H.J. Kropelin T.F. Hoekman J. et al.Drug-induced reduction in albuminuria is associated with subsequent renoprotection: a meta-analysis.J Am Soc Nephrol. 2015; 26: 2055-2064Crossref PubMed Scopus (187) Google Scholar Sodium glucose cotransporter-2 (SGLT-2) inhibitors are increasingly used because of important effects on risk of cardiovascular disease and CKD progression.3Staplin N. Roddick A.J. Emberson J. et al.Net effects of sodium-glucose co-transporter-2 inhibition in different patient groups: a meta-analysis of large placebo-controlled randomized trials.EClinicalMedicine. 2021; 41101163Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Under normoglycemic and modest hyperglycemic conditions, SGLT-2 inhibitors increase urinary glucose excretion to ∼50–80 g/d, and to >100 g/d in people with diabetes and glomerular hyperfiltration. This can equate to urinary glucose concentrations up to ∼500 mmol/L. High serum glucose is known to interfere with the Jaffe reaction used to measure serum creatinine, with bias exceeding 10% with glucose concentrations >20 mmol/L in some assays.4Syme N.R. Stevens K. Stirling C. et al.Clinical and analytical impact of moving from Jaffe to enzymatic serum creatinine methodology.JALM. 2020; 5: 631-642Google Scholar This problem can be circumvented by use of enzymatic creatinine assays,4Syme N.R. Stevens K. Stirling C. et al.Clinical and analytical impact of moving from Jaffe to enzymatic serum creatinine methodology.JALM. 2020; 5: 631-642Google Scholar,5Çuhadar S. Köseoğlu M. Çinpolat Y. et al.The effect of extremely high glucose concentrations on 21 routine chemistry and thyroid Abbott assays: interference study.Biochem Med (Zagreb). 2016; 26: 53-60Crossref PubMed Scopus (4) Google Scholar but such assays are more expensive and use is often low, even in high-income countries (only ∼50% of UK clinical laboratories use enzymatic methods).6UK NEQAS BQUrine chemistries external quality assurance programme, distribution 186.https://ukneqas.org.uk/Date accessed: May 22, 2022Google Scholar Like in serum, glucose may interfere with urinary creatinine measurement, and the potentially high levels of glycosuria resulting from SGLT-2 inhibitor use could cause biases that have important clinical implications.7Heerspink H.J.L. Stefánsson B.V. Correa-Rotter R. et al.Dapagliflozin in patients with chronic kidney disease.N Engl J Med. 2020; 383: 1436-1446Crossref PubMed Scopus (1865) Google Scholar We aimed to quantify this potential bias using laboratory interference studies in urine samples from patients with CKD spiked with a range of glucose concentrations intended to represent the range expected in patients taking SGLT-2 inhibitors. Laboratory and statistical methods are provided in the Supplement (with assay repeatability measurements in Supplementary Table S1). We studied urine samples from 370 patients with CKD: 37 (10%) samples with endogenous glycosuria ≥5.6 mmol/L were excluded, leaving 333 participants' samples for analyses. Median (interquartile range) uACR was 63 (17–150) mg/mmol, with 30 (9%), 72 (22%), and 231 (69%) with uACRs of <3, ≥3 to <30, and ≥30 mg/mmol, respectively (Supplementary Table S2). Median (interquartile range) urinary glucose concentration before spiking with glucose was 0.33 (0.33–0.57) mmol/L. There was no evidence that spiking with glucose had any effect on urinary albumin measurements at 28 or 111 mmol/L glucose concentration, but a 0.5% bias emerged at 333 mmol/ L (Supplementary Figure S1). There was no bias for enzymatic creatinine measurements (Figure 1a). Consequently, overall, there was only a small bias in log uACR measurement when an enzymatic method was used and when urine glucose concentration was 333 mmol/L (Figure 2a).Figure 2Bland-Altman plots for urinary albumin-to-creatinine ratio (uACR), by glucose concentration. Mean bias and 95% confidence intervals (CIs) are shown by dashed and solid gray lines. The 95% limits of agreement are shown as solid black lines. For log-transformed variables, mean bias values have been back-transformed onto the original scale to give a percentage difference.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For the Jaffe creatinine method, however, the presence of glucose caused a bias that resulted in substantial overestimation of urinary creatinine at the lowest creatinine concentrations and a small underestimate at high urinary creatinine concentrations (Figure 1b). This bias was not importantly different across the range of levels of albuminuria (Supplementary Figure S2). The net bias resulting from glucose interference was, on average, to underestimate uACR across the range of albumiuria studied. The Bland-Altman plots in Figure 1b show increasingly steep regression line slopes with higher glucose concentration, indicating increasing bias with higher glucose concentrations. In this cohort, the presence of 28 mmol/L of glucose in the urine resulted in a –1.5% mean bias in uACR (95% confidence interval, –1.9% to –1.1%), which increased to –2.5% (95% confidence interval, –3.2% to –1.9%) at a glucose concentration of 333 mmol/L (Figure 2b). Bias was largest at low creatinine concentrations (i.e., in dilute urine; Supplementary Figure S3). Scatterplots of paired creatinine and uACR measurements are provided in Supplementary Figures S4 and S5. In this particular CKD cohort, where taking SGLT-2 inhibitors was simulated with glucose spiking, interference from the highest level of glucose concentration led to 5.1% (17/333) and 1.8% (6/333) of samples having uACR underestimates of ≥10% and ≥20%, respectively (Supplementary Table S3). We also estimated absolute and percentage change in uACR for different hypothetical levels of uACR. Among the 4% (14/333) of participants with a urine creatinine <2.5 mmol/L, the presence of 28 mmol/L of urinary glucose caused a bias of –5.2% to –7.0% (depending on level of uACR). This bias increased to –10.1% to –13.0% at a concentration of 333 mmol/L. In comparison, biases were all <1.0% for those with urinary creatinine of ≥5 mmol/L (Supplementary Table S4). Results using untransformed values of albumin and uACR are provided in Supplementary Figures S6 and S7. Jaffe assays are commonly used to measure creatinine, and the presence of glycosuria in the range expected to result from use of SGLT-2 inhibitors causes a biased underestimate of uACR when such an assay is used. This bias increases progressively with higher urinary glucose concentrations, and particularly affects dilute urine samples (i.e., urinary creatinine <2.5 mmol/L).5Çuhadar S. Köseoğlu M. Çinpolat Y. et al.The effect of extremely high glucose concentrations on 21 routine chemistry and thyroid Abbott assays: interference study.Biochem Med (Zagreb). 2016; 26: 53-60Crossref PubMed Scopus (4) Google Scholar Any underestimation of uACR by glucose interference creates a positive bias for any observed reduction in uACR in serial uACR measurements. In patients with CKD stages 3 to 4 in this study, high urinary glucose concentration resulted in an ∼10% underestimate of uACR among those with dilute urine. In contrast, enzymatic methods were almost unaffected. Such a level of bias is arguably unacceptable if it alters decisions made by clinicians unaware of the interference. An overestimate of the reduction in albuminuria observed after starting an SGLT-2 inhibitor could, for example, result in a decision not to start other proven kidney disease–modifying treatments or make a patient ineligible for a treatment reserved for people with a certain level of albuminuria. This bias also has implications for analyses from historical and design of future clinical trials. A 30% reduction in geometric mean uACR has been suggested as a meaningful and valid surrogate of treatments for progressive CKD.8Heerspink H.J.L. Greene T. Tighiouart H. et al.Change in albuminuria as a surrogate endpoint for progression of kidney disease: a meta-analysis of treatment effects in randomised clinical trials.Lancet Diabetes Endocrinol. 2019; 7: 128-139Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar Although urinary glucose resulting from SGLT-2 inhibition is unlikely to interfere with Jaffe assays sufficiently to result in a ≥30% change, it could result in overestimated or misleading claims of beneficial effects on uACR. In a review of the published literature, we were only able to identify type of urine assay used in 1 of 11 large placebo-controlled SGLT-2 inhibitor trials and none of 4 intensive versus standard glycemic control trials (Supplementary Table S5).9Zoungas S. Arima H. Gerstein H.C. et al.Effects of intensive glucose control on microvascular outcomes in patients with type 2 diabetes: a meta-analysis of individual participant data from randomised controlled trials.Lancet Diabetes Endocrinol. 2017; 5: 431-437Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar Given the increasing use of SGLT-2 inhibitors in clinical practice, we suggest uACR measured using Jaffe creatinine assays should be avoided, where possible. Alternatively, where resource limitations preclude use of more expensive enzymatic methods, assay type should be reported with results. Methods traceable to the international standard not affected by glycosuria should be used in trials assessing effects of interventions on albuminuria when such interventions could modify glycosuria. The Clinical Trial Service Unit and Epidemiological Studies Unit have a staff policy of not accepting honoraria or other payments from the pharmaceutical industry, except for the reimbursement of costs to participate in scientific meetings (www.ctsu.ox.ac.uk). All authors report a grant to their institution from Boehringer Ingelheim to conduct the EMPA-KIDNEY (the Study of Heart and Kidney Protection with Empagliflozin) trial. Data used in this publication are available in line with the policy and procedures described at: https://www.ndph.ox.ac.uk/data-access. For further information, contact the corresponding author. This work used samples from the UK HARP-III (the United Kingdom Heart and Renal Protection-III) trial, which was designed, conducted, and analyzed by the Medical Research Council-Population Health Research Unit at the University of Oxford, which is part of the Clinical Trial Service Unit and Epidemiological Studies Unit (CTSU), Nuffield Department of Population Health. The University of Oxford was the sponsor for the trial, which was funded by a grant to the University of Oxford from Novartis (the manufacturer of sacubitril/valsartan). The funder had no involvement in the study conduct, analysis, or decision to submit this manuscript for publication. This study was supported by the Medical Research Council (MC_UU_00017/3) and the National Institute for Health Research Clinical Research Network. WGH was supported by a Medical Research Council Kidney Research UK Professor David Kerr Clinician Scientist Award (MR/R007764/1). DC, WGH, and MH conceived the study. DC, TA, SC, SM, and MH designed the interference studies. RH, PKJ, WGH, MJL, and CB collected the data. DC and TA supervised the laboratory analyses. DC, PKJ, WGH, and NS specified the statistical analyses, which were performed by RJS. DC, PKJ, RJS, MH, and WGH drafted the manuscript, which was reviewed and edited for content by all authors. Download .docx (.63 MB) Help with docx files Supplementary File (Word) Supplementary Methods. Supplementary Table S1. Assay repeatability coefficient of variation determined from measuring quality control material over 20 days, with 2 runs per day and 2 replicates per run. Supplementary Table S2. Baseline characteristics. Supplementary Table S3. Percentage of samples with reduction in urinary albumin-to-creatinine ratio (uACR) due to glucose spiking. Supplementary Table S4. Absolute and percentage change in urinary albumin-to-creatinine ratio (uACR) due to glucose interference of urine Jaffe assays by hypothetical levels of uACR. Supplementary Table S5. Methods of albuminuria measurement in other clinical trials. Supplementary Figure S1. Bland-Altman plots for albumin, by glucose concentration. Supplementary Figure S2. Association between creatinine and the difference in urinary albumin-to-creatinine ratio (uACR) before and after adding glucose to a Jaffe assay, by glucose level and uACR at randomization. Supplementary Figure S3. Association between mean creatinine and the difference in urinary albumin-to-creatinine ratio (uACR) before and after adding glucose, by assay method and glucose concentration. Supplementary Figure S4. Creatinine before and after adding glucose, by glucose concentration. Supplementary Figure S5. Urinary albumin-to-creatinine ratio (uACR) before and after adding glucose, by glucose concentration. Supplementary Figure S6. Bland-Altman plots for urinary albumin-to-creatinine ratio (uACR) (untransformed), by glucose concentration. Supplementary Figure S7. Bland-Altman plots for albumin (untransformed), by glucose concentration. Supplementary References. in this issueKidney InternationalVol. 103Issue 4PreviewThe Jaffe reaction is the most commonly used test to measure creatinine levels. High levels of glucose can interfere with Jaffe, causing it to overestimate creatinine. Because the use of sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) is increasing dramatically in patients with chronic kidney disease, Chapman et al. studied whether the glycosuria seen with SGLT2i affects urine creatinine measurement. They found that increasing concentrations of urine glucose, within the range commonly induced by SGLT2 inhibition, did increase urine creatinine, especially in more dilute samples. Full-Text PDF Glucosuria and interference of urinary albumin-to-creatinine ratioKidney InternationalVol. 104Issue 1PreviewWe read with interest the article by Chapman et al.1 regarding the glucosuria interference on the urine albumin-to-creatinine ratio. The authors criticize "the" Jaffe method for assaying creatinine in urine, despite the fact that Jaffe never described a method for measuring creatinine.2 There are several Jaffe assays with their own interference pattern caused by pseudochromogens. Unfortunately, Chapman et al. did not mention the details of their creatinine assay. Urinary creatinine concentrations are generally 2 orders of magnitude higher than the plasma/serum creatinine values, necessitating a 100-fold specimen dilution, making the assay less susceptible to analytical interference. Full-Text PDF The authors replyKidney InternationalVol. 104Issue 1PreviewWe thank Delanghe et al.1 for their interest in our study.2 They suggest the analytical bias in our study may be due to specific compensations with the Jaffe assay we used. The urine method used in our study is a widely available kinetic Jaffe method without mathematical compensation that uses a calibrator traceable to National Institute of Standards and Technology standard reference material (NIST SRM) 3667 (product numbers are provided in the Supplementary Methods2). Interference by glucose using this method has been assessed as <3% by the manufacturer up to 167 mmol/L (3000 mg/dl); however, they only tested one pooled urine sample. Full-Text PDF